DHA affects ISKNV-induced autophagy flux via the AMPK/mTOR/ULK1 and BECN1/SQSTM1 axes, limiting viral replication in fish cells.

Abstract

Docosahexaenoic acid (DHA) plays important roles in organisms, including in the structure and liquidity of cell membranes, anti-oxidation, and anti-inflammation. There has been very little work on the influence of DHA on autophagic flux regulation, especially with respect to large DNA viruses. In this paper, we demonstrate that ISKNV can trigger host autophagy flux via the AMPK/mTOR/ULK1 and Beclin-1(BECN1)/p62(SQSTM1) axes, which are suppressed by DHA in GF-1 cells. We found that DHA (10 μM) treatment could enhance host cell viability by up to 85 % on day 5 post-infection. Then, in an autophagosome assay, we found that the DHA-treated groups exhibited reductions of 50 % in MDC-positive signals upon ISKNV infection in GF-1 cells. In the autophagic marker LC3-II, we found that ISKNV could induce autophagy-relative markers, an effect which was also suppressed by DHA treatment 3 and 5 days post-infection. Through studies on molecular autophagy flux, we found that ISKNV could induce autophagy pathways via AMPK/mTOR/ULK1 signaling and was also involved via phosphorylation of Serine 555 and Serine 757 of ULK-1 on days 3 and 5 post-infection in GF-1 cells. Furthermore, we found that ISKNV can dramatically induce BECN1/p62(SQSTM1) signaling, but these two signaling pathways were all modulated by DHA treatment on days 3 and 5 post-infection. Finally, DHA treatment was found to suppress viral gene expression of DdDp and MCP, which correlated with 20-fold and 5.3-fold differences in viral titers on days 3 and 5, respectively, with ISKNV infection in GF-1 cells. Taken together, these results suggest that DHA treatment can affect autophagy flux via both AMPK/mTOR/ULK1 and BECN1/SQSTM1 signaling, which correlates with reduced viral gene expression and viral replication in GF-1 cells. This finding may provide insight into large DNA virus infections and therapeutic treatment.